Background
Sarcoidosis is a systemic granulomatous disease of unknown cause. A genetic predisposition is suggested by familial histories and the risk for the disease multiplied by 80 in monozygotic twins of index cases [
1,
2]. Genome-wide association studies and candidate gene-driven investigations identified several genetic risk foci for sarcoidosis, including butyrophilin-like 2 gene [
3‐
5], Annexin 11 [
6], a locus on chromosome 11q13.1 [
7], several loci in the HLA region [
8] and in the IL23/Th17 signalling [
9].
NOD2 mutations have been evidenced in granulomatous diseases, including Blau syndrome (BS), early onset sarcoidosis (EOS) and Crohn’s disease (CD). NOD2 is a cytosolic protein involved in sensing microbial cell wall components and regulating inflammatory processes by activating NF-κB [
10]. A deficit in sensing bacteria in monocytes/macrophages might result in an exaggerated inflammatory response by the adaptive immune system. Thus
NOD2 mutations are considered to play a major role in the pathogenesis of granulomatous diseases [
11,
12]. Variants may concern different NOD2 domains, NACHT domain for BS and EOS and LRR domain for CD [
11,
13]. NACHT domain variants enhanced NOD2 activity, whereas LRR domain variants reduced NOD2.
Although scientific evidence was provided indicating that the NOD2 signalling pathway was involved in sarcoidosis pathogenesis, selection of patients with confirmed sarcoidosis from the Sarcoidosis Genetic Analysis study population [
14] and the ACCESS study [
15] with or without joint and skin involvement showed no evidence of
NOD2 mutation [
16]. Up to now, no study has evidenced any
NOD2 mutation in sarcoidosis with a late typical presentation. The role of the
NOD2 gene in sarcoidosis was associated with increase susceptibility for developing sarcoidosis [
17,
18].
The SARCFAM project is a French national project on familial sarcoidosis which allowed the recruitment of more than 180 families with at least two first-degree affected members [
19]. Screening of nine sarcoidosis by WES has provided extensive and complex data which are included in a larger program of the SARCFAM project. Among this series, a subset of 10 families gathered more than 3 patients. One of them referred as “X” was of particular interest due to the identification of NOD2 variants, including the G908R mutation that has been described in Crohn’s disease. Identification of the
NOD2 variant in the LRR domain (G908R in exon 8) was confirmed by Sanger sequencing.
In the present study, we report sarcoidosis presentation and history, NOD2 profile and NF-κB and cytokine production in blood monocytes in affected patients of the family X and to compare genetic and NF-κB and cytokine profiles with members of the family unaffected by sarcoidosis and safe controls.
Discussion
To our knowledge, this is the first time that the NOD2 2722G > C (rs2066845) variant substituting a Glycine to Arginine at codon 908 in exon 8 in the leucine rich repeat (LRR) domain of the NOD2 protein is reported in a case of familial sarcoidosis with late and typical presentation of the disease. This variant was observed in an unaffected relative, suggesting that other genetic and/or epigenetic factors may contribute to the occurrence of the disease. Indeed, WES analysis discriminated affected versus unaffected individuals by three putative pathogenic SNP variants in the IL17RA, KALRN and EPHA2 genes.
NOD2 is essential in regulating both inflammatory and immunologic homeostasis.
In silico pathogenic evaluation (p) with the SIFT (
p = 0.01) and POLYPHENv2 (PSIC score difference = 2.407) suggest a strong disturbing effect on the primary amino acid sequence raising the question of a significant impact in sarcoidosis pathogenesis. The role of the G908R mutation of the
NOD2 gene in sarcoidosis is controversial either with increase susceptibility for developing sarcoidosis [
17,
18] or a minor role of the
2722G > C variant in the pathogenesis of sarcoidosis [
22‐
24]. In the present case of familial sarcoidosis, 3 out of 4 patients with the variant
2722G > C developed sarcoidosis, while the other unaffected offspring did not carry the variant. Observation of a family member with NOD2 variant but no sarcoidosis is similar to the numerous individuals free of any disease, particularly of CD despite carrying the same variant, indicating that gene/environmental interactions and possibly gene/gene interactions (epistasis) are also essential [
25]. Such association of sarcoidosis with the
NOD2 2722G > C variant is certainly a very rare finding. It was not observed in Bello’s paper focused on adult patients with sarcoidosis involving eyes, skin and joints as in EOS and BS [
16]. In our nine other families with at least 3 members affected, none was associated to any
NOD2 variant. Among our family collection, families with 3 or more affected members (
n = 10) represent probably a small subset among sarcoidosis families with probably specific genetic predisposal or shared environmental factors.
It is of particular interest to observe the
NOD2 2722G > C variant associated with a very typical presentation of sarcoidosis, all patients showing bilateral hilar lymphadenopathy and lymphatic distribution of lung micronodules distinct from EOS when lung involvement was present [
26]. By contrast evidence of lung infiltration is very rare in EOS with a very different radiologic pattern and no lymphadenopathy [
11]. Another question is the impact of NOD2 variant on the outcome which was characterized by a very long course in all patients, a condition usual in CD and perhaps associated to persistent abnormalities of microbiota induced by the variant. Indeed, deletion of
Nod2 in mice reduced bacterial activity in the gut, promoting an increased susceptibility to colonization by both the commensal microbiota and pathogenic bacteria, thus triggering intestinal inflammation [
27]. In addition,
Nod2 deletion impairs autophagy in macrophages allowing intracellular survival of bacteria delaying bacterial clearance [
28]. In our family, any confusion with CD either as main diagnosis or as comorbidity was ruled out by the absence of suggestive history of digestive tract disease particularly in one patient who had two colonoscopic examinations.
Both gain of function NOD2 mutant alleles like in BS and EOS and loss of function NOD2 mutant alleles like in most common CD and probably in our family with sarcoidosis might induce autoinflammatory disorders [
29,
30]. During sarcoidosis, NF-κB is activated in site of organ involved [
31‐
33]. In BS and EOS, gain of function NOD2 variants are associated with NF-κB activation. By contrast, we observed NFκB downregulation in our untreated patient as in CD patients with the
NOD2 2722G > C variant. These data are supported by
in vitro experiments showing that NFκB activity is reduced at baseline and in response to MDP in HEK293T cells expressing the
NOD2 2722G > C variant [
34‐
36]. Interestingly, NF-κB activity was upregulated in response to MDP in family X treated patients as well as the unaffected carrier of the variant, similarly to normal controls suggesting that treatments efficient against sarcoidosis activity may restore NF-κB activity altered by the presence of the
NOD2 2722G > C variant. Indeed, patients treated by glucocorticoids responded well to the treatment, indicating that they were not refractory to corticoids [
37] by comparison to Crohn disease patients [
38]. Although increased NF-κB activity in patients with corticoids is obviously in conflict with what could be expected with anti-inflammatory drugs, previous studies showed that glucocorticoids could potentially contribute to pro-inflammatory activation by inducing the expression of the Macrophage migration inhibitory factor [
39]. By contrast, in response to LPS stimulation, NF-κB activity was maintained in the untreated patient IB to similar levels observed in family X treated patients and normal controls, indicating that stimulation of NF-κB through NOD2- independent pathways remains possible. NF-κB is an important transcription factor for genes encoding cytokines like
IL-8 and
TNF-A, this last one being a corner stone in sarcoidosis pathogenesis. In patient IB, despite a decrease in NF-κB activity,
IL-8 and
TNF-A mRNA levels were increased at baseline and in stimulated conditions, indicating a chronic pro-inflammatory status in macrophages. As in Crohn disease patients with the
NOD2 2722G > C variant, patient IB led to an apparent paradox: reduced NF-κB transactivating activity associated with more inflammation. Recently Strober et al... showed that in mice, activation of NF-κB in response to ligands to toll-like receptor 2 (TLR-2) is down-regulated by NOD2 [
40]. In the absence of an efficient form of NOD2 the TLR signaling pathway is maintained, leading to increased production of inflammatory cytokines and the development of pathogenesis. Other data suggest that virulence and pathogens factors may trigger the NOD1/NOD2 signaling pathway indirectly by activating Rac1 [
41]. Rac1 has been shown to upregulate NF-κB activity in a PAK1 (p21-activated kinase) dependent manner by stimulating the nuclear translocation of p65 subunit of NF-κB [
42]. In addition, Hedl et al showed that acute stimulation of NOD2 triggered in human macrophages the secretion of anti-inflammatory cytokines, including IL-10 and IL1ra [
43]. Thus, we could hypothesize that in
NOD2 2722G > C patients, an imbalance occurs in favour of a pro-inflammatory phenotype.
One of the major issue was to find any genetic data which might discriminate family X members expressing clinically the disease from the others. Remarkably, in addition to
NOD2 2722G > C, three single nucleotide variants for the
IL17RA,
KALRN and
EPHA2 genes were found in the family X patients with sarcoidosis. By comparison, as shown in Table
3, screening of the nine families by WES analysis showed the presence of single deleterious variants in a total of 7 families out of 9, one of them (
EPHA2 – rs139787163) being common in two distinct families despite a very low minor allele frequency (MAF = 0.000346).
The
IL17RA 958 T > C variant modifies the amino acid sequence in the extracellular domain of the IL17RA, possibly altering its function. IL17RA (interleukin 17A receptor) is capable to form a heterodimer complex to bind both interleukin 17A and IL-25. IL17RA plays a pathogenic role in many inflammatory and autoimmune diseases, including rheumatoid arthritis, psoriasis,
Candida albicans infection and Crohn disease [
44‐
46]. Activation of IL17RA leads to induction of expression of inflammatory cytokines such as CXCL1, CXCL8/IL8 and IL6 through the trans-activation of NF-κB, AP-1 and C/EBPβ. Involvement of IL17 signalling was shown in sarcoidosis [
47]. Notably, IL-17–producing T cells are increased in peripheral blood and lungs of subjects with sarcoidosis compared with controls [
48]. In addition, IL-17A plays a major role in granuloma formation in response to mycobacterial infections in mice [
49,
50]. Likewise Th17 cells have been implicated in the development of Crohn’s disease [
51]. Notably, McGovern et al... showed that IL17RA genetic variants increased susceptibility to inflammatory bowel disease pathogenesis and demonstrated the cumulative risk of IL17RA variants with genes of the IL23/IL17 pathway in the development of CD [
46]. Interestingly, Kurdy et al demonstrated that IL17 is directly regulated at the transcriptional level by a complex including Rac1, Tiam1 and RORχt (RAR-related orphan receptor gamma), the master transcription and differentiation factor of Th17 cells [
52].
The
EPHA2 2875G > A variant is located within the conserved cytoplasmic domain of the receptor. This variant was associated with age-related cortical cataract [
53‐
56]. Erythropoietin-producing hepatoma (Eph) receptors are a family of receptor tyrosine kinases that can bind ephrin ligands. EphA receptors are expressed on CD4
+ and CD8
+ T cells, dendritic cells (DCs), and Langherans cells [
57,
58] and are involved in T cell function [
59]. Eph receptors are involved in axon guidance, vascularization, tissue assembly, and cell adhesion and migration [
60‐
63]. Khounlothm et al... showed that EphA receptors play a role in the pathogenesis of
M. tuberculosis infection by reducing the migration of T cells and DCs to the site of infection, producing an environment that favours bacterial persistence [
64]. Activated EPHA2 interacts with Rho family GEF in endosomes and play a role in ephrin-dependant Rac1 activation [
65]. Interestingly, variants for EPH receptor family members have been associated with inflammatory bowel diseases. Hafner et al showed an increased expression of Eph-B2 that increased epithelial cell mobility in the intestinal epithelium of Morbus Crohn patients [
66].
The KALRN 28C > T variant is a non-sense variant targeting Kalirin, a multidomain guanine nucleotide exchange factor (GEF) for small GTP-binding proteins of the Rho family. Multiple kalirin isoforms containing different combinations of functional domains are predominantly expressed in brain, except KALRN 9 that is more widely expressed [
67]. Kalirin is known to be involved in active remodeling of synapses and dendritic maturation in early development [
68]. Kalirin was also shown to play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity [
69]. In smooth muscle cells, Kalirin interacts with Rac1, promoting SMC migration and proliferation [
70]. Thus, it appears that both EPHA2 and KALRN are implicated in dendritic cell maturation and migration. Dendritic cells have been implicated in sarcoidosis [
71]. Mathew et al. showed that myeloids DCs function was reduced in sarcoidosis patients, possibly contributing to susceptibility and persistence of the chronic inflammation [
72]. Interestingly in family X, member IIC who carries the
NOD2 2722G > C variant only
, did not develop sarcoidosis. These data suggest that the
NOD2 2722G > C variant was not sufficient to trigger pulmonary sarcoidosis by itself and it is the presence of other variants (
IL17RA 958 T > C, EPHA2 2875G > A and
KALRN 28C > T) that contribute to the pathology in patients with pulmonary sarcoidosis of family X, perhaps by enhancing the development and chronicity of pulmonary sarcoidosis in family X.
Taken together, our data suggest a functional and pathogenic link between EPHA2, KALRN and IL17RA in the occurrence of the disease of family X sarcoidosis patients, acting in addition to the NOD2 functional defect.